scholarly journals Moderate SMFs attenuate bone loss in mice by promoting directional osteogenic differentiation of BMSCs

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Guilin Chen ◽  
Yujuan Zhuo ◽  
Bo Tao ◽  
Qian Liu ◽  
Wenlong Shang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Bioinformatics Analysis ◽  
Therapeutic Option ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Osteoblastic Differentiation ◽  
Dependent Manner ◽  
Bone Mass Loss ◽  
Lineage Differentiation ◽  
All Trans Retinoic Acid

Abstract Background Osteoporosis is a common metabolic bone disease without effective treatment. Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to differentiate into multiple cell types. Increased adipogenic differentiation or reduced osteogenic differentiation of BMSCs might lead to osteoporosis. Whether static magnetic fields (SMFs) might influence the adipo-osteogenic differentiation balance of BMSCs remains unknown. Methods The effects of SMFs on lineage differentiation of BMSCs and development of osteoporosis were determined by various biochemical (RT-PCR and Western blot), morphological (staining and optical microscopy), and micro-CT assays. Bioinformatics analysis was also used to explore the signaling pathways. Results In this study, we found that SMFs (0.2–0.6 T) inhibited the adipogenic differentiation of BMSCs but promoted their osteoblastic differentiation in an intensity-dependent manner. Whole genomic RNA-seq and bioinformatics analysis revealed that SMF (0.6 T) decreased the PPARγ-mediated gene expression but increased the RUNX2-mediated gene transcription in BMSCs. Moreover, SMFs markedly alleviated bone mass loss induced by either dexamethasone or all-trans retinoic acid in mice. Conclusions Taken together, our results suggested that SMF-based magnetotherapy might serve as an adjunctive therapeutic option for patients with osteoporosis.

scholarly journals Effect of local anesthetics on viability and differentiation of various adult stem/progenitor cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Young Hoon Kim ◽  
Ga Young Park ◽  
Nechama Rabinovitch ◽  
Solaiman Tarafder ◽  
Chang H. Lee
Keyword(s):  
Progenitor Cells ◽  
Local Anesthetics ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Dose Effect ◽  
Specific Gene ◽  
Gene Expressions ◽  
Differentiation Capacity ◽  
Matrix Deposition ◽  
Lineage Differentiation

Abstract Background Local anesthetics (LAs) are widely used to control pain during various clinical treatments. One of the side effects of LAs, cytotoxicity, has been investigated in various cells including stem/progenitor cells. However, our understanding of the effects of LAs on the differentiation capacity of stem/progenitor cells still remains limited. Therefore, a comparative study was conducted to investigate the effects of multiple LAs on viability and multi-lineage differentiation of stem/progenitor cells that originated from various adult tissues. Method Multiple types of stem/progenitor cells, including bone marrow mesenchymal stem/progenitor cells (MSCs), dental pulp stem/progenitor cells (DPSCs), periodontal ligament stem/progenitor cells (PDLSCs), and tendon-derived stem/progenitor cells, were either obtained from a commercial provider or isolated from adult human donors. Lidocaine (LD) and bupivacaine (BP) at various doses (1×, 0.75×, 0.5×, and 0.25× of each physiological dose) were applied to the different stem/progenitor cells for an hour, followed by induction of fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation. Live/dead and MTT assays were performed at 24 h after the LD or BP treatment. At 2 weeks, qRT-PCR was conducted to evaluate the gene expressions associated with differentiation. After 4 weeks, multiple biochemical staining was performed to evaluate matrix deposition. Results At 24 h after LD or BP treatment, 1× and 0.75× physiological doses of LD and BP showed significant cytotoxicity in all the tested adult stem/progenitor cells. At 0.5×, BP resulted in higher viability than the same dose LD, with variance between cell types. Overall, the gene expressions associated with fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation were attenuated in LD or BP pre-treated stem/progenitor cells, with notable dose-effect and dependence on types. In contrast, certain doses of LD and/or BP were found to increase specific gene expression, depending on the cell types. Conclusion Our data suggest that LAs such as LD and BP affect not only the viability but also the differentiation capacity of adult stem/progenitor cells from various anatomical sites. This study sheds light on stem cell applications for tissue regeneration in which isolation and transplantation of stem cells frequently involve LA administration.

scholarly journals Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

2021 ◽  
Author(s):  
Kannan Govindaraj ◽  
Sakshi Khurana ◽  
Marcel Karperien ◽  
Janine Nicole Post
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transcription Factor ◽  
Single Cell ◽  
Adipogenic Differentiation ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Transcriptional Dynamics

The master transcription factor SOX9 is a key player during chondrocyte differentiation, cartilage development, homeostasis and disease. Modulation of SOX9 and its target gene expression is essential during chondrogenic, osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs). However, lack of sufficient knowledge about the signaling interplay during differentiation remains one of the main reasons preventing successful application of hMSCs in regenerative medicine. We previously showed that Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP) can be used to study SOX9 dynamics at the single cell level. We showed that changes in SOX9 dynamics are linked to its transcriptional activity. Here, we investigated SOX9 dynamics during differentiation of hMSCs into the chondrogenic, osteogenic and adipogenic lineages. We show that there are clusters of cells in hMSCs with distinct SOX9 dynamics, indicating that there are a number of subpopulations present in the heterogeneous hMSCs. SOX9 dynamics data at the single cell resolution revealed novel insights about its activity in these subpopulations (cell types). In addition, the response of SOX9 to differentiation stimuli varied in these subpopulations. Moreover, we identified donor specific differences in the number of cells per cluster in undifferentiated hMSCs, and this correlated to their differentiation potential.

Acute Myeloid Leukemia (AML) Cells Alter the Bone Marrow Microenvironment By Inducing Osteogenic and Suppressing Adipogenic Differentiation of MSCs through BMP-RUNX2-CTGF Mediated Mechanisms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2403-2403
Author(s):  
Venkata Lokesh Battula ◽  
Phuong M Le ◽  
Jeffrey Sun ◽  
Hong Mu ◽  
Teresa Mc.Queen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Cell Growth ◽  
Osteogenic Differentiation ◽  
Research Funding ◽  
Adipogenic Differentiation ◽  
Time Dependent ◽  
Dependent Manner ◽  
Osteoprogenitor Cells

Abstract Osteoprogenitor cells are a critical component of the bone marrow microenvironment (BME), support AML cell growth (Raaijmakers et al., Nature, 2010); however the mechanism has not been fully elucidated. Here, we hypothesize that AML cells induce osteogenic differentiation in mesenchymal stem/stromal cells to gain growth advantage. We have isolated age matched (between 40-70 years) bone marrow derived MSCs from AML patients (AML-MSC, n=20) and normal (N-MSC, n=10) individuals and analyzed their cell growth, cell surface phenotype and multi-lineage differentiation. AML-MSCs are phenotypically different with larger cell surface area and lower cell growth compared to N-MSCs. The average doubling time of AML-MSCs is 52±8hrs compared to 34±6hours for N-MSCs (p<0.01). Phenotypic and enzyme activity studies revealed that tissue non-specific alkaline phosphatase (TNAP), a protein that is highly expressed in naïve MSCs and osteoprogenitor cells and osteoblasts, is expressed 10-14 fold higher in AML-compared to N-MSCs (p<0.01). As TNAP is associated with osteogenic lineage, we next assessed the expression of osteogenic markers including RUNX-2, osteopontin, TNAP and osterix. Interestingly, these genes were up-regulated by 5-10-fold in AML-MSCs compared to N-MSCs. To validate these observations, N-MSCs were cultured with OCI-AML3 cell derived conditioned medium (CM) for 3-5 days and then induced to osteogenic or adipogenic differentiation. As expected, alkaline phosphatase enzyme activity and Alizarine Red S staining was twice as high in MSCs cultured with AML-CM compared to MSCs cultured in regular cell culture medium. In addition, we found expression of osteogenic genes including RUNX-2, osteopontin, TNAP and osterix 3-4 fold upregulated in MSCs cultured with AML-CM compared to control MSCs. These data indicate that AML cells prime MSCs to undergo osteogenic differentiation. Adipocyte differentiation was assessed by Oil-Red O staining for lipid droplets and revealed a >95% reduction (p<0.001) in the number of mature adipocytes in AML-MSCs compared to N-MSCs. Gene expression analysis by qRT-PCR revealed that adipogenic markers including aP2, lipoprotein lipase and PPARγ were down-regulated 10-20 fold in AML-MSCs compared to N-MSCs in a time-dependent manner suggesting that AML-MSCs lack the ability to differentiate into adipocytes. To evaluate these findings in-vivo, we developed a Human Bone Marrow Implant (HBMI) mouse model by subcutaneous implantation of human femur derived bone pieces into NSG mice. In this model, mice bearing HBMI with leukemia expressed higher osteogenic related proteins compared to mice without leukemia. Importantly, multispectral image analysis revealed 5-10 fold higher osteogenic activity in AML patient BM-biopsies compared to normal bones. Bone morphogenic proteins (BMP) are the most essential factors during osteogenic differentiation and new bone formation in humans. We therefore treated MSCs with AML cell-derived conditioned medium, and observed a 6-8 fold increase in pSmad1/5 levels in N-MSCs in a time dependent manner. In addition, AML induced pSmad1/5 up-regulation was inhibited when MSCs were treated with BMP-type1 receptor specific inhibitor LDN-212854, in a dose dependent manner. In addition, inhibition of BMP signaling also inhibited AML-induced osteogenic differentiation in MSCs. Mechanistic studies revealed that bone morphogenetic proteins (BMPs) derived from AML cells induce connective tissue growth factor (CTGF) expression in MSCs. When transplanted in Col1a2-CTGF transgenic mice, AML cells engrafted faster compared to wild type mice suggesting that CTGF enhances AML cell growth. Our data suggest that AML cells induce osteogenic differentiation in MSCs and receive growth advantage through BMP-CTGF signaling. Inhibition of these pathways may reduce leukemia propagation and improve AML patient survival. Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

scholarly journals Differential Ability of Somatic Stem Cells

2009 ◽  
Vol 18 (5-6) ◽  
pp. 581-590 ◽  
Author(s):  
Koichi Oishi ◽  
Hirofumi Noguchi ◽  
Hiroshi Yukawa ◽  
Shuji Hayashi
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Es Cells ◽  
Adipogenic Differentiation ◽  
Embryonic Stem ◽  
Cell Types ◽  
Somatic Stem Cells ◽  
Serum Free ◽  
Pancreatic Stem Cells ◽  
A Cell

Somatic stem cells can be isolated from a variety of sources. Although some studies have suggested that somatic stem cells may represent a cell population that is very similar to embryonic stem (ES) cells, it remains unclear whether somatic stem cells retain the potential to differentiate into any cell type derived from the three germ layers. In this study, we investigated the transdifferentiation potential of somatic stem cells using adipose tissue-derived stem/progenitor cells (ASCs; mesodermal stem cells) and pancreatic stem cells (endodermal stem cells). Previous reports from other groups describe the protocol that has been used to differentiate ASCs or mesenchymal stem cells (MSCs) in bone marrow into insulin-producing cells. Induction 1: ASCs were cultured for 3 days in ultra-low attachment plates under serum-free conditions. Induction 2: ASCs were cultured for 24 h with L-DMEM, and reinduced with serum-free H-DMEM for another 10 h. Unlike previous reports, we did not get ASCs to express any pancreas-specific genes, including insulin-1 or insulin-2. Pancreatic stem cells were induced to differentiate into adipo/osteogenic by the following protocols. Induction protocol 1: ACSs were cultured for 7 days with medium containing indometacin, dexamethasone, hydrocortisone, and insulin for adipogenic differentiation. Induction protocol 2: The cells were cultured for 7 days with medium containing dexamethasone, ascorbate-2-phosphate, and β-glycerophosphate for osteogenic differentiation. Although these approaches have been widely used for adipo/osteogenic differentiation from MSCs, adipo/osteogenic differentiation from pancreatic stem cells was not observed. These data suggest that it is not easy for somatic stem cells to transdifferentiate into other germ cell types, at least, under these conditions.

scholarly journals Resveratrol Induces the Osteogenic Differentiation via MiR-320c by Targeting Runx2 and Is Involved in the Adipogenic differentiation of BMSCs

2020 ◽  
Author(s):  
Jilong Zou ◽  
Jianyang Du ◽  
Hualei Tu ◽  
Hongjun Chen ◽  
Kai Cong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Target Gene ◽  
Target Genes ◽  
Adipogenic Differentiation ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Matrix Mineralization

Abstract Background Bone marrow mesenchymal stem cells (BMSCs) are multipotent progenitor cells and have been widely used in clinical therapies due to their multiple pluripotency. Recent publications have found that resveratrol (RSVL) could promote the proliferation and differentiation of mesenchymal stem cells; however, the underlying molecular mechanism of RSVL-induced BMSCs osteogenic differentiation needs to be fully elucidated. The aim of this study was to investigate the function of miRNAs in RSVL-treated BMSCs and its effects on the osteogenic differentiation of BMSCs. Methods BMSCs were cultured and treated with different concentrations of RSVL. After osteogenic differentiation for 20 days, ALP staining was performed to evaluate the ALP activity of BMSCs. And ARS staining was used to detect the matrix mineralization deposition of BMSCs. After adipogenic differentiation for 20 days, adipogenic differentiation was determined by ORO staining for lipid droplets. Quantitative real-time polymerase chain reaction analysis was performed to assess the expression level of target genes. Bioinformatics analysis and luciferase reporter assay was ultilized to examine the relationship between miR-320c and its target gene. Western blot assay was used to analyze the protein expression level of target gene. Results Our results demonstrated that RSVL could promote the osteogenic differentiation and suppressed the adipogenic differentiation of BMSCs in a dose-dependent manner. Besides, a novel regulatory axis containing miR-320c and its target Runx2 was found during the differentiation process of BMSCs under RSVL treatment. Overexpression of miR-320c inhibited the osteogenic differentiation, while knockdown of miR-320c promoted the osteogenic differentiation of BMSCs. In contrast, overexpression of miR-320c accelerated the adipogenic differentiation, while knockdown of miR-320c restrained the adipogenic differentiation of BMSCs. Our results confirm that Runx2 was the directly target of miR-320c in RSVL-promoted osteogenic differentiation of BMSCs. Conclusions The present study revealed that miR-320c might possess the potentials as a novel clinical target for medical intervention to regulate the biological functions of RSVL in BMSCs.

scholarly journals Identification of Co-Expression Network Correlated With Different Periods of Adipogenic and Osteogenic Differentiation of BMSCs by Weighted Gene Co-Expression Network Analysis (WGCNA)

2021 ◽  
Author(s):  
Yu Liu ◽  
Markus Tingart ◽  
Sophie Lecouturier ◽  
Jianzhang Li ◽  
Jörg Eschweiler
Keyword(s):  
Osteogenic Differentiation ◽  
Expression Pattern ◽  
Ribosome Biogenesis ◽  
Acid Metabolism ◽  
Bioinformatics Analysis ◽  
Adipogenic Differentiation ◽  
Gene Expression Pattern ◽  
Cell Staining ◽  
Tnf Α ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: The differentiation of bone marrow mesenchymal stem cells (BMSCs) is a complex and dynamic process. The gene expression pattern and mechanism of different periods of adipogenic and osteogenic differentiation remain unclear. Additionally the inaction between these two lineages determination requires further exploration. Results: Five modules that are most significantly associated with osteogenic or adipogenic differentiation of BMSCs were selected for further investigation. Biological terms, such as ribosome biogenesis, TNF-α signaling pathway, glucose import, fatty acid metabolism along with hub transcript factors, such as PPARG, YY1, and hub miRNAs, such as hsa-mir-26b-5p were enriched in different modules. The expression pattern of 6 hub genes, ADIPOQ, FABP4, SLC7A5, SELPLG, BIRC3, and KLHL30 were validated by RT-qPCR. In the end, cell staining experiments extended the findings of bioinformatics analysis.Conclusion: This study identified the key genes, biological functions, and regulators of each time point of adipogenic and osteogenic differentiation of BMSCs and provided novel evidence and ideas for further research on the differentiation of BMSCs.

314 ADIPOSE- AND BONE MARROW-DERIVED MESENCHYMAL STEM CELLS PRESENT LARGE SIMILARITIES IN TRANSCRIPTOME PRIOR TO AND DURING ADIPOGENIC AND OSTEOGENIC DIFFERENTIATION

2011 ◽  
Vol 23 (1) ◽  
pp. 253 ◽  
Author(s):  
E. Monaco ◽  
M. Bionaz ◽  
A. Lima ◽  
W. L. Hurley ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Antigen Presentation ◽  
Osteogenic Differentiation ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Significant Enrichment ◽  
Post Hoc

Previous data support adipose-derived stem cells as an alternative to bone marrow as a source of adult stem cells for therapeutic purposes. The aim of the present study was to directly compare the transcriptome of adipose-derived (ADSC) and bone marrow-derived (BMSC) mesenchymal stem cells prior to differentiation and during in vitro osteogenic and adipogenic differentiation. The ADSC and BMSC were harvested from 3 adult pigs and differentiated in vitro into adipocytes and osteocytes for up to 4 weeks. Prior to differentiation and at differentiation day 2, 7, and 21, cells were harvested and RNA extracted for transcriptomics analysis by a 13 263 oligo 70-mers array (Sus scrofa AROS V1.0 with extension; Operon). Data were normalized by Lowess and statistical analysis was run using ANOVA with Benjamini-Hochberg false discovery rate (FDR) correction. Data mining was carried out using Ingenuity Pathway Analysis and David. Using an FDR of <0.05 for overall tissue effect and a post-hoc correction of P < 0.001, we observed 65 differentially expressed genes (DEG) between ADSC and BMSC before starting differentiation (0.66% of unique genes in the array). Functional analysis uncovered significant enrichment of extracellular matrix genes with direct roles in cell adhesion, migration, movement, and morphology. When the interaction cell type × differentiation × time was assessed, we observed >2 000 DEG with an FDR <0.05. This large number was mostly due to time effects. When pair-wise comparisons between cell types for each time point during the same differentiation were performed (post-hoc P < 0.001), we observed a strikingly low number of DEG. The number of DEG was lower between cell types in osteogenic (<100 DEG) compared with adipogenic (<200 DEG) differentiation. We observed significant enrichment (FDR-corrected P-value cut-off <0.05) of functions related to metabolism, antigen presentation, angiogenesis, and cell cycle in both differentiation conditions. We also observed an overall greater induction of the enriched functions in ADSC and a decrease in BMSC during adipogenic differentiation and the opposite during osteogenic differentiation except for metabolism, which appeared to be larger in ADSC in all cases. Among the significant enriched functions of DEG between the 2 differentiations, we observed enrichment of genes involved in metabolism, cell death, cell-to-cell signalling, and antigen presentation in ADSC during adipogenic compared with osteogenic differentiation. In BMSC we observed enrichment of functions related to cell death, antigen presentation, and lipid metabolism in osteogenic v. adipogenic differentiation. Overall data uncovered a high similarity at the transcriptional level between ADSC and BMSC both prior to differentiation and during differentiation. Those data support ADSC being particularly similar to BMSC. This work was support by the Illinois Regenerative Medicine Institute (IDPH # 63080017).

Mycophenolic Acid Inhibits the Proliferation and Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Guanosine Depletion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1454-1454 ◽  
Author(s):  
Weijie Cao ◽  
Lizhen Liu ◽  
Xiaoyu Lai ◽  
Xiaohong Yu ◽  
He Huang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Adipogenic Differentiation ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Dependent Manner ◽  
Oil Red O Staining ◽  
Oil Red O

Abstract Abstract 1454 Poster Board I-477 Introduction Mycophenolate mofetil is now widely used in transplantation as a potent immunosuppressant, whose active metabolite is mycophenolic acid (MPA). MPA inhibits de novo purine biosynthesis by reversible, noncompetitive inhibition of inosine monophosphate dehydrogenase (IMPDH). The inhibition of IMPDH in lymphocytes reduces intracellular guanine nucleotide pools, thus arrests lymphocytes proliferation. Recently investigators reported the antiproliferative effects of MPA on fibroblasts, smooth muscle cells and endothelial cells, but there is no reports of the effects of MPA on human bone marrow-derived mesenchymal stem cells (MSCs). Here we examined the effects of MPA on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells. Methods Bone marrow aspirates were obtained from healthy volunteers after informed consent, and MSCs were expanded from bone marrow mononuclear cells by discarding non-adherent cells. For proliferation and survival assays, MSCs were treated with MPA at the concentration of 1μM, 10μM, 50μM, and 100μM. Cell proliferation was analyzed using CCK-8 method (Dojindo). Cell viability was assessed by trypan blue exclusion. Apoptosis was detected by PI/Annexin V assay kit (Invitrogen). To assess the effects of MPA on MSCs differentiation, osteogenic differentiation and adipogenic differentiation were induced in the presence of MPA. For the detection of osteogenic differentiation, the deposited minerals was stained with silver by the method of von Kossa and Ca2+ contents was quantified with calcium colorimetric assay kit (Biovision). Adipogenic differentiation was analyzed by Oil Red O staining and Oil Red O staining extraction. Results In the range of 1μM to 100μM, MPA caused a significant subdued proliferation rate of MSCs in a concentration- and time-dependent manner. After 7d of incubation with MPA at the concentration of 1μM, 10μM, 50μM, and 100μM, the proliferation rate was reduced to 65.33±11.03%, 24±3.74%, 15.33±4.03%, and 15.33±6.94% respectively (P<0.01). Adding guanosine (100μM) to the culture restored the proliferation rate (P<0.01) indicating that MPA exerted antiproliferative effects by guanosine depletion. Trypan blue staining showed that there was no statistically significant difference in the ratio of living cells between MPA treated cells and the control group (P>0.05), and PI/Annexin V staining showed no apoptosis induce by MPA. Von Kossa stainnging indicated that treatment with MPA reduced Ca2+ deposition during osteogenic differentiation of MSCs, and Ca2+ quantification further confirmed that MPA inhibited osteogenic differentiation in a concentration-dependent manner. Ca2+ quantification was 78.43±12.79 μg/well and 22.8±6.58 μg/well respectively at the concentration of 10μM and 100μM of MPA, which were significantly lower than the control group(118.33±12.50ug/well, P<0.05). Oil Red O staining and Quantification of lipid contents showed that MPA had no effect on lipid production during adipogenic differentiation. Conclusion Our study demonstrated that MPA inhibited the proliferation of MSCs by guanosine depletion, and also inhibited the osteogenic differentiation in a concentration-dependent manner. However, MPA had no impact on adipogenic differentiation in vitro. Disclosures No relevant conflicts of interest to declare.

scholarly journals Influence of Human Cytomegalovirus Glycoprotein O Polymorphism on the Inhibitory Effect of Soluble Forms of Trimer- and Pentamer-Specific Entry Receptors

2020 ◽  
Vol 94 (14) ◽  
Author(s):  
Nadja Brait ◽  
Tanja Stögerer ◽  
Julia Kalser ◽  
Barbara Adler ◽  
Ines Kunz ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Hcmv Infection ◽  
Therapeutic Option ◽  
Cell Types ◽  
Complete Inhibition ◽  
Dependent Manner ◽  
Response Curves ◽  
Entry Receptor

ABSTRACT Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO trimer and gH/gL/UL128-131 pentamer, are important for cell-free HCMV entry. While soluble NRP2-Fc (sNRP2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble platelet-derived growth factor receptor α-Fc (sPDGFRα-Fc) interacts with gO, thereby inhibiting infection of all cell types. Since gO is the most variable subunit, we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, and GT5 sequences in the bacterial artificial chromosome (BAC) TB40-BAC4-luc background (where luc is luciferase). All mutants were tested for fibroblast and epithelial cell infectivity, for virion content of gB, gH, and gO, and for infection inhibition by sPDGFRα-Fc and sNRP2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in gB and gH levels in mutant virions. All gO GT mutants except recombinant gO GT1c/3 displayed a nearly complete inhibition at 1.25 μg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%), and all experienced complete inhibition on fibroblasts (≥99%). While gO GT replacement did not influence sNRP2-Fc inhibition at 1.25 μg/ml on epithelial cells (97% to 99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblast inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope of >1.0), sNRP2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0, suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2 in a cell type-dependent manner. IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism, as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the amounts of trimer in virions and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information for the future development of anti-HCMV therapeutic compounds based on sPDGFRα.

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